5' PHOSPHATE MIMICS

• US 20120157511A1
• Filed: 07/07/2010
• Published: 06/21/2012
• Est. Priority Date: 07/07/2009
• Status: Active Grant

• Current Assignee: Alnylam Pharmaceuticals Incorporated
• Original Assignee: Alnylam Pharmaceuticals Incorporated
• Inventors: Manoharan, Muthiah, Rajeev, Kallanthottathil G., Zlatev, Ivan, Prhavc, Marija
• Granted Patent: US 8,927,513 B2
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/11 containing cyclic phosphate
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/213 containing cyclic phosphate
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/11 Antisense

Abstract

The present invention provides nucleosides and oligonucleotides comprising a 5′ phosphate mimics of formula (IVc) or (Vc). One aspect of the present invention relates to modified nucleosides and oligonucleotides comprising such dinucleotide of formula (Ia). Another aspect of the invention relates to a method of inhibiting the expression of a gene in call, the method comprising (a) contacting an oligonucleotide of the invention with the cell; and (b) maintaining the cell from step (a) for a time sufficient to obtain degradation of the mRNA of the target gene.

Claims

1. A nucleoside represented by formula (II):

2. The nucleoside of claim 1, represented by formula (III):

3. The nucleoside of claim 1, represented by formula (IV):

4. The nucleoside of claim 1, where V and W₁ are taken together with the atom to which they are bonded to form a nucleoside represented by formula (V):

5. An oligonucleotide comprising a nucleoside of formula (IIa):

6. The oligonucleotide of claim 5, wherein the nucleoside is represented by formula (IIIa):

7. The oligonucleotide of claim 5, wherein the nucleoside is represented by formula (IVa):

8. The oligonucleotide of claim 5, wherein the nucleoside is represented by formula (Va):

9. The oligonucleotide of claim 5, wherein said oligonucleotide comprises at least one non-phosphodiester backbone linkage.

10. The oligonucleotide of claim 9, wherein the non-phosphodiester of said non-phosphodiester backbone linkage is selected from a group consisting of phosphorothioate, phosphorodithioate, alkyl-phosphonate, and phosphoramidate.

11. The oligonucleotide of claim 9, wherein the non-phosphodiester backbone linkage is placed between the first two nucleosides at the 5′

- end and/or between the first two nucleosides at the 3′

- end.

12. The oligonucleotide of claim 5, wherein said oligonucleotide comprises at least one ligand conjugate.

13. The oligonucleotide of claim 5, wherein said oligonucleotide is a double-stranded oligonucleotide comprising a first strand and a second strand.

14. The oligonucleotide of claim 13, wherein the nucleoside of formula (IIa) is present either in the first strand or the second strand of the oligonucleotide.

15. The oligonucleotide of claim 14, wherein said first strand is the sense strand.

16. The oligonucleotide of claim 14, wherein said second strand is the antisense strand.

17. The oligonucleotide of claim 13, wherein the dinucleotide of formula (IIa) is present in both strands of the oligonucleotide.

18. The oligonucleotide of claim 5, wherein said oligonucleotide has a hairpin structure.

19. The oligonucleotide of claim 5, wherein said oligonucleotide is an antisense, an antagomir, a microRNA, a siRNA, a pre-microRNA, a supermir, an antimir, a ribozyme, a decoy oligonucleotide, RNA activator, an U1 adaptor, an immunestimmulatory oligonucleotide or an aptamer.

20. The oligonucleotide of claim 19, wherein said oligonucleotide is a single-stranded siRNA.

21. A method of inhibiting the expression of a target gene in a cell, the method comprising

a. contacting an oligonucleotide of claim 5 with the cell;

and

b. maintaining the cell from step (a) for a time sufficient to obtain degradation of the mRNA of the target gene.

22. The method of claim 21, wherein, the target gene is selected from the group consisting of Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu gene, topoisomerase I gene, topoisomerase II alpha gene, p73 gene, p21(WAF1/CIP1) gene, p27(KIP1) gene, PPM1D gene, RAS gene, caveolin I gene, MIB I gene, MTAI gene, M68 gene, mutations in tumor suppressor genes, p53 tumor suppressor gene, and combinations thereof.

23. A pharmaceutical composition, comprising the oligonucleotide of claim 5 and a pharmaceutically acceptable excipient.

24. A method of silencing a disease-causing gene, comprising administering to a subject in need thereof the pharmaceutical composition of claim 23.

25. The method of claim 24, wherein the disease-causing gene is selected from the group consisting of Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu gene, topoisomerase I gene, topoisomerase II alpha gene, p73 gene, p21(WAF1/CIP1) gene, p27(KIP1) gene, PPM1D gene, RAS gene, caveolin I gene, MIB I gene, MTAI gene, M68 gene, mutations in tumor suppressor genes, and p53 tumor suppressor gene.